Single cell analyses from our lab and others suggest the processes involved in steroid receptor-based transcription are tightly regulated with respect to spatiotemporal dynamics within living cells. Using fluorescently-tagged estrogen receptor-alpha (ER) and several coregulators in cell culture model systems, we will test the hypothesis that regulation of both dynamics and compartmentalization are necessary for transcriptional activation. This test will include examination of ER function prior to, during, and after transcription using highly quantitative imaging I approaches. Real-time visualization of transcription dynamics will be explored in three specific aims: Aim I will further characterize ER/coregulator organization, dynamics and turnover, in living and fixed cells, using spectrally I compatible "GFP fusions during ligand/non-ligand dependent activation. Time-lapse and multi-spectral imaging, and photobleaching approaches will be augmented by pulse-chase experiments and ultrastructural comparison direct from live cells using FlAsH epitope tagging; FRET studies will provide visual protein-protein interaction assessment throughout the nucleus. Aim II will utilize lac- and tet-repressor fusions and integrated lac- and tet-operator DNA arrays to examine ER/coregulator interactions and focus upon local chromatin effects with a' fluorescent reporter. Aim III will focus upon new cells containing integrated transcription units by using a series of ER-responsive prolactin (PRL) promoters that allow visualization of the integration sites and provide biosensor readout of transcription activity. Real-time photobleaching data with new PRL arrays reveal ER and coregulator are highly dynamic (tl/2 = approximately seconds) when binding the integrated promoter. Direct spatiotemporal examination of receptor-coregulator interactions are now possible and will test the link between ER activation, chromatin modulation and transcription; concurrent chromatin immunoprecipitation studies will be performed to assess dynamics from a biochemical approach. These novel approaches will provide vital information regarding the function of ER, and mechanisms it uses at its site of action, within the living cell, allowing multiplex analyses of several aspects of ER function simultaneously. For the first time, mechanistic and quantitative scrutiny of early events (seconds/minutes) of receptor activation will be possible leading to and directly following transcription, and should lead to development of novel strategies for drug therapies, specifically in hormone-responsive cancers.